Method used for detecting HLA-B*5801 alleles

A technology of HLA-B and alleles, applied in the field of alleles, can solve the problems of difficult interpretation of primer results, inconvenient result interpretation, increased workload, etc., and achieve accurate and reliable detection results, easy operation, and less interference factors.

Active Publication Date: 2014-01-01
安徽同科生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using 24 pairs of primers will not only increase the workload, but will also cause inconvenience in the interpretation of the results. This ambiguity will appear in up to 20 heterozygous allele combinations

Method used

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  • Method used for detecting HLA-B*5801 alleles
  • Method used for detecting HLA-B*5801 alleles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Detection of HLA-B*5801 alleles by PCR-SSP method

[0070] 1. Extract DNA:

[0071] After collecting venous blood (2ml) in vacuum blood collection tubes anticoagulated with ethylenediaminetetraacetic acid (EDTA) according to conventional methods, DNA was extracted using the QIAamp DNA Extract Kit (Qiagen, Germany);

[0072] 2. Design sequence-specific primers and internal reference primers:

[0073]Design three pairs of specific primers for HLA-B*5801 in the concentrated area of ​​HLA-B gene polymorphism sites, the first upstream primer F1: 5'-CCGGGTCCGAGATCCGTCT-3' (sequence 1), the second upstream primer F2 : 5'-GGGCCGGAGTATTGGGATG-3' (SEQ ID NO: 2), the third upstream primer F3: 5'-accgagagaacctgcggat-3' (SEQ ID NO: 3), the universal downstream primer R: 5'-gccatacatcctctggatga-3' (SEQ ID NO: 4); , using the Primer 3 software (http: / / frodo.wi.mit.edu / ) to design internal reference primers on the β-globin gene, the upstream primer Globin-F: 5'- AGTCAGGGCAG...

Embodiment 2

[0079] Example 2 Detection of HLA-B*5801 alleles by Taqman probe method

[0080] 1. Extract DNA:

[0081] After collecting venous blood (2ml) in vacuum blood collection tubes anticoagulated with ethylenediaminetetraacetic acid (EDTA) according to conventional methods, DNA was extracted using the QIAamp DNA Extract Kit (Qiagen, Germany);

[0082] 2. Design primers and probes:

[0083] Design specific primers for HLA-B*5801 in the polymorphic site concentrated region, upstream primer F2: 5'-GGGCCGGAGTATTGGGATG-3' (SEQ ID NO: 2), downstream primer R: 5'-gccatacatcctctggatga-3' (SEQ ID NO: 4) , and two supporting fluorescent probes: probe1: 5'-CY5-TCCGAGATCCGCCTCCCTGAGGCC-BHQ2-3' (SEQ ID NO: 7) and probe2: 5'-JOE- ACCGAGAGAACCTGCGGATCGCGCTCC-BHQ2-3' (SEQ ID NO: 8); Internal reference primers were designed on the globin gene, upstream primer Globin-F: 5'- AGTCAGGGCAGAGCCATCTA-3' (SEQ ID NO: 5), downstream primer Globin-R: 5'-TTAGGGTTGCCCATAACAGC-3' (SEQ ID NO: 6), and supporting ...

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Abstract

The invention belongs to the field of pharmacogenomics and genetic diagnosis, and relates to a method used for detecting HLA-B*5801 alleles. The method comprises following steps: a DNA sample to be detected is taken, three pairs of specific primers and a pair of internal primers are taken, amplification of DNA segments is realized by using sequence specific primer method, and then the results of the amplification are analyzed by agarose gel electrophoresis; or sample DNA is extracted, a pair of specific primers, a pair of internal primers and three fluorescence probes are taken, amplification of DNA segments is realized by Taqman probe method using a fluorescence ration PCR instrument, and then the amplification curve is analyzed so as to obtain results. Results analysis methods such as agarose gel electrophoresis, high resolution melting curve and SYBRGreen fluorogenic quantitative PCR are employed in the method. The method has advantages of speediness, convenience, flexibility, high resolution and no contamination; is suitable for detection of HLA-B*5801 alleles in samples such as peripheral blood and hair; and can be used for determining the probability of severe skin adverse reaction of patients with gout or hyperuricemia caused by taking of allopurinol.

Description

technical field [0001] The invention belongs to the fields of pharmacogenomics and gene diagnosis, and specifically relates to a method for detecting HLA-B*5801 alleles and an application thereof. Background technique [0002] With the sharp increase in the intake of high-protein, high-purine, and high-calorie foods in people's diets, as well as the increase in alcohol consumption (especially beer) and the decrease in activity, the number of patients with obesity and metabolic syndrome has increased, making The incidence of concomitant hyperuricemia and primary gout is on the rise. Studies have shown that gout is caused by abnormal metabolism of purine substances, excessive uric acid production or poor excretion of uric acid, resulting in elevated uric acid in the blood, and deposition of urate crystals in the synovium, synovial bursa, cartilage and other tissues. Recurrent inflammatory disease, which is characterized by limited joint movement, heat, pain, and swelling, acc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2561/101C12Q2600/106C12Q2600/156C12Q2531/113
Inventor 邹和建张心菊于波关明张炯骆肖群张伟万峻吕元
Owner 安徽同科生物科技有限公司
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